Transgenic mice have become a powerful research tool for the study of gene expression and protein function in mammals. Once a transgenic line is established, itself a demanding task, it would be of great benefit to immortalize the sperm of males of the line to maintain the line with minimum space and cost requirements. Cryopreservation offers an approach to sperm immortalization. But while sperm cropreservation has been successful with many mammalian species, it has not been so with mouse sperm. The overall objective of the proposed research is to define the cryobiology of mouse sperm, to utilize this definition to formulate optimum sperm cryopreservation protocols, and to demonstrate that live cryopreserved sperm can transmit the transgene. Freezing and thawing of sperm cells subjects the cells to osmotic forces that cause water efflux from the cell during freezing, such that intracellular freezing is avoided, and water influx during thawing. The resulting stress on the plasma membrane induced by the intracellular volume changes may damage it. This is one mode of cryodamage; the other is lethal intracellular ice formation, which occurs if water efflux is too slow. The rate of water movement across the plasma membrane, defined by its hydraulic conductivity, Lp, must be optimal to avoid the two extremes. Determination of Lp as a function of temperature is thus crucial to sperm cryobiology. A cryoprotectant, usually glycerol, is required to minimize cryodamage by inhibiting lethal intracellular freezing. Its intracellular concentration is also affected by osmotic forces, so determination of the plasma membrane's permeability coefficient to glycerol, Pg, is essential to sperm cryobiology. The first specific aim derives from the need to measure these parameters in order to attain the objective of the research. Aim I is to determine for mouse sperm: Lp, Pg, and their temperature dependences; effects of cooling and warming rates, glycerol concentration, an medium osmolality on intracellular ice formation; membrane phase transition temperatures; and the effects of freezing the sperm suspension to a glass. The standard flow cytometry methods and new methods using fluorescent probes will be used to determine parameters and to assay effects. Aim 2 is to formulate an optimized cryopreservation protocol based on the sperm cryobiology parameters and verify the fertilizing capacity of the live sperm recovered from freeze-thaw. In vitro fertilization methods currently in use will be used in this study. Aim 3 is to verify that cryopreserved sperm do transmit transgenes. Two transgenic lines with readily assayed transgene expression phenotypes will he used for this purpose. Both in vitro fertilization followed by embryo transfer and artificial insemination will be used to produce the progeny to be tested for the transgene.